Vasopressin formulations for use in treatment of hypotension

ABSTRACT

Provided herein are peptide formulations comprising polymers as stabilizing agents. The peptide formulations can be more stable for prolonged periods of time at temperatures higher than room temperature when formulated with the polymers. The polymers used in the present invention can decrease the degradation of the constituent peptides of the peptide formulations.

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No.14/610,499, filed Jan. 30, 2015, which is incorporated herein byreference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 20, 2015, isnamed 47956-702.301_SL.txt and is 5120 bytes in size.

BACKGROUND

Vasopressin is a potent endogenous hormone, responsible for maintainingplasma osmolality and volume in most mammals. Vasopressin can be usedclinically in the treatment of sepsis and cardiac conditions, and in theelevation of patient's suffering from low blood pressure. Currentformulations of vasopressin require refrigeration for maintenance orreconstitution of lyophilized powders due to vasopressin's poorlong-term stability.

SUMMARY OF THE INVENTION

In some embodiments, the invention provides a pharmaceutical compositioncomprising, in a unit dosage form: a) from about 0.01 mg/mL to about0.07 mg/mL of vasopressin, or a pharmaceutically-acceptable saltthereof; and b) a polymeric pharmaceutically-acceptable excipient in anamount that is from about 1% to about 10% by mass of the unit dosageform or the pharmaceutically-acceptable salt thereof, wherein the unitdosage form exhibits from about 5% to about 10% less degradation of thevasopressin or the pharmaceutically-acceptable salt thereof afterstorage for about 1 week at about 60° C. than does a corresponding unitdosage form, wherein the corresponding unit dosage form consistsessentially of: A) vasopressin, or a pharmaceutically-acceptable saltthereof; and B) a buffer having acidic pH.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a chromatogram of a diluent used in vasopressin assay.

FIG. 2 is a chromatogram of a sensitivity solution used in a vasopressinassay.

FIG. 3 is a chromatogram of an impurity marker solution used in avasopressin assay.

FIG. 4 is a zoomed-in depiction of the chromatogram in FIG. 3.

FIG. 5 is a chromatogram of a vasopressin standard solution.

FIG. 6 is a chromatogram of a sample vasopressin preparation.

FIG. 7 is a UV spectrum of a vasopressin sample.

FIG. 8 is a UV spectrum of a vasopressin standard.

FIG. 9 plots vasopressin stability across a range of pH as determinedexperimentally.

FIG. 10 illustrates the effects of various stabilizers on vasopressinstability.

DETAILED DESCRIPTION Vasopressin and Peptides of the Invention

Vasopressin, a peptide hormone, acts to regulate water retention in thebody and is a neurotransmitter that controls circadian rhythm,thermoregulation, and adrenocorticotrophic hormone (ACTH) release.Vasopressin is synthesized as a pro-hormone in neurosecretory cells ofthe hypothalamus, and is subsequently transported to the pituitary glandfor storage. Vasopressin is released upon detection of hyperosmolalityin the plasma, which can be due to dehydration of the body. Uponrelease, vasopressin increases the permeability of collecting ducts inthe kidney to reduce renal excretion of water. The decrease in renalexcretion of water leads to an increase in water retention of the bodyand an increase in blood volume. At higher concentrations, vasopressinraises blood pressure by inducing vasoconstriction.

Vasopressin acts through various receptors in the body including, forexample, the V1, V2, V3, and oxytocin-type (OTR) receptors. The V1receptors occur on vascular smooth muscle cells, and the major effect ofvasopressin action on the V1 receptor is the induction ofvasoconstriction via an increase of intracellular calcium. V2 receptorsoccur on the collecting ducts and the distal tubule of the kidney. V2receptors play a role in detection of plasma volume and osmolality. V3receptors occur in the pituitary gland and can cause ACTH release uponvasopressin binding. OTRs can be found on the myometrium and vascularsmooth muscle. Engagement of OTRs via vasopressin leads to an increaseof intracellular calcium and vasoconstriction.

Vasopressin is a nonapeptide, illustrated below (SEQ ID NO. 1):

At neutral to acidic pH, the two basic groups of vasopressin, theN-terminal cysteine, and the arginine at position eight, are protonated,and can each carry an acetate counterion. The amide groups of theN-terminal glycine, the glutamine at position four, and the asparagineat position five, are susceptible to modification when stored asclinical formulations, such as unit dosage forms. The glycine,glutamine, and asparagine residues can undergo deamidation to yield theparent carboxylic acid and several degradation products as detailed inEXAMPLE 1 and TABLE 1 below.

Deamidation is a peptide modification during which an amide group isremoved from an amino acid, and can be associated with proteindegradation, apoptosis, and other regulatory functions within the cell.Deamidation of asparagine and glutamine residues can occur in vitro andin vivo, and can lead to perturbation of the structure and function ofthe affected proteins. The susceptibility to deamidation can depend onprimary sequence of the protein, three-dimensional structure of theprotein, and solution properties including, for example, pH,temperature, ionic strength, and buffer ions. Deamidation can becatalyzed by acidic conditions. Under physiological conditions,deamidation of asparagine occurs via the formation of a five-memberedsuccinimide ring intermediate by a nucleophilic attack of the nitrogenatom in the following peptide bond on the carbonyl group of theasparagine side chain. Acetylation is a peptide modification whereby anacetyl group is introduced into an amino acid, such as on the N-terminusof the peptide.

Vasopressin can also form dimers in solution and in vivo. Thevasopressin dimers can occur through the formation of disulfide bridgesthat bind a pair of vasopressin monomers together. The dimers can formbetween two parallel or anti-parallel chains of vasopressin.

Vasopressin and associated degradation products or peptides are listedin TABLE 1 below. All amino acids are L-stereoisomers unless otherwisedenoted.

TABLE 1 Name Sequence SEQ ID NO. Vasopressin (AVP; arginineCYFQNCPRG-NH₂ 1 vasopressin) Gly9-vasopressin (Gly9-AVP) CYFQNCPRG 2Asp5-vasopressin (Asp5-AVP) CYFQDCPRG-NH₂ 3 Glu4-vasopressin (Glu4-AVP)CYFENCPRG-NH₂ 4 Glu4Gly9-vasopressin (Glu4Gly9- CYFENCPRG 5 AVP)AcetylAsp5-vasopressin Ac-CYFQDCPRG-NH₂ 6 (AcetylAsp5-AVP)Acetyl-vasopressin (Acetyl-AVP) Ac-CYFQNCPRG-NH₂ 7 His2-vasopressin(His2-AVP) CHFQNCPRG-NH₂ 8 Leu7-vasopressin (Leu7-AVP) CYFQNCLRG-NH₂ 9D-Asn-vasopressin (DAsn-AVP) CYFQ(D-Asn)CPRG-NH₂ 10 D-Cys1-vasopressin(D-Cys)YFQNCPRG-NH₂ 11 D-Tyr-vasopressin C(D-Tyr)FQNCPRG-NH₂ 12D-Phe-vasopressin CY(D-Phe)QNCPRG-NH₂ 13 D-Gln-vasopressinCYF(D-Gln)NCPRG-NH₂ 14 D-Cys6-vasopressin CYFQN(D-cys)PRG-NH₂ 15D-Pro-vasopressin CYFQNC(D-pro)RG-NH₂ 16 D-Arg-vasopressinCYFQNCP(D-Arg)G-NH₂ 17

Therapeutic Uses.

A formulation of vasopressin can be used to regulate plasma osmolalityand volume and conditions related to the same in a subject. Vasopressincan be used to modulate blood pressure in a subject, and can beindicated in a subject who is hypotensive despite treatment with fluidand catecholamines.

Vasopressin can be used in the treatment of, for example, vasodilatoryshock, post-cardiotomy shock, sepsis, septic shock, cranial diabetesinsipidus, polyuria, nocturia, polydypsia, bleeding disorders, VonWillebrand disease, haemophilia, platelet disorders, cardiac arrest,liver disease, liver failure, hypovolemia, hemorrhage, oesophagealvariceal haemorrhage, hypertension, pulmonary hypertension, renaldisease, polycystic kidney disease, blood loss, injury, hypotension,meniere disease, uterine myomas, brain injury, mood disorder.Formulations of vasopressin can be administered to a subject undergoing,for example, surgery or hysterectomy.

Plasma osmolality is a measure of the plasma's electrolyte-water balanceand can be indicative of blood volume and hydration of a subject. Normalplasma osmolality in a healthy human subject range from about 275milliosmoles/kg to about 295 milliosmoles/kg. High plasma osmolalitylevels can be due to, for example, diabetes insipidus, hyperglycemia,uremia, hypernatremia, stroke, and dehydration. Low plasma osmolalitycan be due to, for example, vasopressin oversecretion, improperfunctioning of the adrenal gland, lung cancer, hyponatremia,hypothyroidism, and over-consumption of water or other fluids.

Septic shock can develop due to an extensive immune response followinginfection and can result in low blood pressure. Causes of sepsis caninclude, for example, gastrointestinal infections, pneumonia,bronchitis, lower respiratory tract infections, kidney infection,urinary tract infections, reproductive system infections, fungalinfections, and viral infections. Risk factors for sepsis include, forexample, age, prior illness, major surgery, long-term hospitalization,diabetes, intravenous drug use, cancer, use of steroidal medications,and long-term use of antibiotics. The symptoms of sepsis can include,for example, cool arms and legs, pale arms and legs, extreme bodytemperatures, chills, light-headedness, decreased urination, rapidbreathing, edema, confusion, elevated heart rate, high blood sugar,metabolic acidosis, respiratory alkalosis, and low blood pressure.

Vasopressin can also be administered to regulate blood pressure in asubject. Blood pressure is the measure of force of blood pushing againstblood vessel walls. Blood pressure is regulated by the nervous andendocrine systems and can be used as an indicator of a subject's health.Chronic high blood pressure is referred to as hypertension, and chroniclow blood pressure is referred to as hypotension. Both hypertension andhypotension can be harmful if left untreated.

Blood pressure can vary from minute to minute and can follow thecircadian rhythm with a predictable pattern over a 24-hour period. Bloodpressure is recorded as a ratio of two numbers: systolic pressure (mmHg), the numerator, is the pressure in the arteries when the heartcontracts, and diastolic pressure (mm Hg), the denominator, is thepressure in the arteries between contractions of the heart. Bloodpressure can be affected by, for example, age, weight, height, sex,exercise, emotional state, sleep, digestion, time of day, smoking,alcohol consumption, salt consumption, stress, genetics, use of oralcontraceptives, and kidney disease.

Blood pressure for a healthy human adult between the ages of 18-65 canrange from about 90/60 to about 120/80. Hypertension can be a bloodpressure reading above about 120/80 and can be classified ashypertensive crisis when there is a spike in blood pressure and bloodpressure readings reach about 180/110 or higher. Hypertensive crisis canbe precipitated by, for example, stroke, myocardial infarction, heartfailure, kidney failure, aortic rupture, drug-drug interactions, andeclampsia. Symptoms of hypertensive crisis can include, for example,shortness of breath, angina, back pain, numbness, weakness, dizziness,confusion, change in vision, nausea, and difficulty speaking.

Vasodilatory shock can be characterized by low arterial blood pressuredue to decreased systemic vascular resistance. Vasodilatory shock canlead to dangerously low blood pressure levels and can be corrected viaadministration of catecholamines or vasopressin formulations.Vasodilatory shock can be caused by, for example, sepsis, nitrogenintoxication, carbon monoxide intoxication, hemorrhagic shock,hypovolemia, heart failure, cyanide poisoning, metformin intoxication,and mitochondrial disease.

Post-cardiotomy shock can occur as a complication of cardiac surgery andcan be characterized by, for example, inability to wean fromcardiopulmonary bypass, poor hemodynamics in the operating room,development of poor hemodynamics post-surgery, and hypotension.

Pharmaceutical Formulations.

Methods for the preparation of compositions comprising the compoundsdescribed herein can include formulating the compounds with one or moreinert, pharmaceutically-acceptable excipients. Liquid compositionsinclude, for example, solutions in which a compound is dissolved,emulsions comprising a compound, or a solution containing liposomes,micelles, or nanoparticles comprising a compound as disclosed herein.These compositions can also contain minor amounts of nontoxic, auxiliarysubstances, such as wetting or emulsifying agents, pH buffering agents,and other pharmaceutically-acceptable additives.

Non-limiting examples of dosage forms suitable for use in the disclosureinclude liquid, elixir, nanosuspension, aqueous or oily suspensions,drops, syrups, and any combination thereof. Non-limiting examples ofpharmaceutically-acceptable excipients suitable for use in thedisclosure include granulating agents, binding agents, lubricatingagents, disintegrating agents, anti-adherents, anti-static agents,surfactants, anti-oxidants, coloring agents, flavouring agents,plasticizers, preservatives, suspending agents, emulsifying agents,plant cellulosic material and spheronization agents, and any combinationthereof.

Vasopressin can be formulated as an aqueous formulation or a lyophilizedpowder, which can be diluted or reconstituted just prior to use. Upondilution or reconstitution, the vasopressin solution can be refrigeratedfor long-term stability for about one day. Room temperature incubationor prolonged refrigeration can lead to the generation of degradationproducts of vasopressin.

In some embodiments, a pharmaceutical composition of the invention canbe formulated for long-term storage of vasopressin at room temperaturein the presence of a suitable pharmaceutically-acceptable excipient. Thepharmaceutically-acceptable excipient can increase the half-life ofvasopressin when stored at any temperature, such as room temperature.The presence of the pharmaceutical excipient can decrease the rate ofdecomposition of vasopressin at any temperature, such as roomtemperature.

In some embodiments, a vasopressin formulation of the inventioncomprises a pharmaceutically-acceptable excipient, and the vasopressinhas a half-life that is at least about 1%, at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 100%, at least about 150%, at least about 200%, at leastabout 250%, at least about 300%, at least about 350%, at least about400%, at least about 450%, at least about 500%, at least about 600%, atleast about 700%, at least about 800%, at least about 900%, or at leastabout 1000% greater than the half-life of vasopressin in a correspondingformulation that lacks the pharmaceutically-acceptable excipient.

In some embodiments, a vasopressin formulation of the invention has ahalf-life at about 0° C. that is no more than about 1%, no more thanabout 5%, no more than about 10%, no more than about 15%, no more thanabout 20%, no more than about 25%, no more than about 30%, no more thanabout 35%, no more than about 40%, no more than about 45%, no more thanabout 50%, no more than about 55%, no more than about 60%, no more thanabout 65%, no more than about 70%, no more than about 75%, no more thanabout 80%, no more than about 85%, no more than about 90%, no more thanabout 95%, no more than about 100%, no more than about 150%, no morethan about 200%, no more than about 250%, no more than about 300%, nomore than about 350%, no more than about 400%, no more than about 450%,no more than about 500%, no more than about 600%, no more than about700%, no more than about 800%, no more than about 900%, or no more thanabout 1000% greater than the half-life of the formulation at anothertemperature, such as room temperature.

The half-life of the compounds of the invention in a formulationdescribed herein at a specified temperature can be, for example, about 1hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours,about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours,about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours,about 60 hours, about 3 days, about 4 days, about 5 days, about 6 days,or about one week.

In some embodiments, a vasopressin formulation of the inventioncomprises an excipient and the vasopressin has a level of decompositionat a specified temperature that is about 1%, about 5%, about 10%, about15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95%, about 100%, about 150%, about200%, about 250%, about 300%, about 350%, about 400%, about 450%, about500%, about 600%, about 700%, about 800%, about 900%, or about 1000%less than the level of decomposition of a formulation of the inventionin the absence of the excipient.

Pharmaceutical compositions of the invention can be used, stored,tested, analyzed or assayed at any suitable temperature. Non-limitingexamples of temperatures include about 0° C., about 1° C., about 2° C.,about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., about8° C., about 9° C., about 10° C., about 11° C., about 12° C., about 13°C., about 14° C., about 15° C., about 16° C., about 17° C., about 18°C., about 19° C., about 20° C., about 21° C., about 22° C., about 23°C., about 24° C., about 25° C., about 26° C., about 27° C., about 28°C., about 29° C., about 30° C., about 31° C., about 32° C., about 33°C., about 34° C., about 35° C., about 36° C., about 37° C., about 38°C., about 39° C., about 40° C., about 41° C., about 42° C., about 43°C., about 44° C., about 45° C., about 46° C., about 47° C., about 48°C., about 49° C., about 50° C., about 51° C., about 52° C., about 53°C., about 54° C., about 55° C., about 56° C., about 57° C., about 58°C., about 59° C., about 60° C., about 61° C., about 62° C., about 63°C., about 64° C., about 65° C., about 66° C., about 67° C., about 68°C., about 69° C., about 70° C., about 71° C., about 72° C., about 73°C., about 74° C., or about 75° C.

Pharmaceutical compositions of the invention can be used, stored,tested, analyzed or assayed at room temperature. The room temperaturecan be, for example, about 20.0° C., about 20.1° C., about 20.2° C.,about 20.3° C., about 20.4° C., about 20.5° C., about 20.6° C., about20.7° C., about 20.8° C., about 20.9° C., about 21.0° C., about 21.1°C., about 21.2° C., about 21.3° C., about 21.4° C., about 21.5° C.,about 21.6° C., about 21.7° C., about 21.8° C., about 21.9° C., about22.0° C., about 22.1° C., about 22.2° C., about 22.3° C., about 22.4°C., about 22.5° C., about 22.6° C., about 22.7° C., about 22.8° C.,about 22.9° C., about 23.0° C., about 23.1° C., about 23.2° C., about23.3° C., about 23.4° C., about 23.5° C., about 23.6° C., about 23.7°C., about 23.8° C., about 23.9° C., about 24.0° C., about 24.1° C.,about 24.2° C., about 24.3° C., about 24.4° C., about 24.5° C., about24.6° C., about 24.7° C., about 24.8° C., about 24.9° C., or about 25.0°C.

A pharmaceutical composition of the disclosure can be a combination ofany pharmaceutical compounds described herein with other chemicalcomponents, such as carriers, stabilizers, diluents, dispersing agents,suspending agents, thickening agents, and/or excipients. Thepharmaceutical composition facilitates administration of the compound toan organism. Pharmaceutical compositions can be administered intherapeutically-effective amounts, for example, intravenous,subcutaneous, intramuscular, transdermal, or parenteral administration.

Pharmaceutical preparations can be formulated for intravenousadministration. The pharmaceutical compositions can be in a formsuitable for parenteral injection as a sterile suspension, solution, oremulsion in oily or aqueous vehicles, and can contain formulation agentssuch as suspending, stabilizing, and/or dispersing agents.Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Suspensions of the active compounds can be prepared as oily injectionsuspensions. Suitable lipophilic solvents or vehicles include fatty oilssuch as sesame oil, or synthetic fatty acid esters, such as ethyl oleateor triglycerides, or liposomes. Aqueous injection suspensions cancontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. The suspensioncan also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions. Alternatively, the active ingredient can be inpowder form for constitution with a suitable vehicle, for example,sterile pyrogen-free water, before use.

Comparison Formulations.

A pharmaceutical composition described herein can be analyzed bycomparison to a reference formulation. A reference formulation can begenerated from any combination of compounds, peptides, excipients,diluents, carriers, and solvents disclosed herein. Any compound,peptide, excipient, diluent, carrier, or solvent used to generate thereference formulation can be present in any percentage, ratio, oramount, for example, those disclosed herein. The reference formulationcan comprise, consist essentially of, or consist of any combination ofany of the foregoing.

A non-limiting example of a comparison formulation comprises, consistsessentially of, or consists of: an amount, such as about 20 Units orabout 0.04 mg, of vasopressin or a pharmaceutically-acceptable saltthereof, an amount, such as about 5 mg, of chlorobutanol (for example,hydrous), an amount, such as about 0.22 mg, of acetic acid or apharmaceutically-acceptable salt thereof or a quantity sufficient tobring pH to about 3.4 to about 3.6, and water as needed. Anothernon-limiting example of a comparison formulation comprises, consistsessentially of, or consists of: vasopressin or apharmaceutically-acceptable salt thereof, chlorobutanol, acetic acid,and a solvent such as water. Another non-limiting example of acomparison formulation comprises, consists essentially of, or consistsof: vasopressin or a pharmaceutically-acceptable salt thereof,chlorobutanol, and a solvent such as water. Another non-limiting exampleof a comparison formulation comprises, consists essentially of, orconsists of: vasopressin or a pharmaceutically-acceptable salt thereof,acetic acid, and a solvent such as water. Another non-limiting exampleof a comparison formulation comprises, consists essentially of, orconsists of: vasopressin or a pharmaceutically-acceptable salt thereofand a solvent such as water. Another non-limiting example of acomparison formulation comprises, consists essentially of, or consistsof: vasopressin or a pharmaceutically-acceptable salt thereof and abuffer having acidic pH, such as pH 3.5 or any buffer or pH describedherein.

Dosage Amounts.

In practicing the methods of treatment or use provided herein,therapeutically-effective amounts of the compounds described herein areadministered in pharmaceutical compositions to a subject having adisease or condition to be treated. A therapeutically-effective amountcan vary widely depending on the severity of the disease, the age andrelative health of the subject, the potency of the compounds used, andother factors. Subjects can be, for example, humans, elderly adults,adults, adolescents, pre-adolescents, children, toddlers, infants, orneonates. A subject can be a patient.

Pharmaceutical compositions of the invention can be formulated in anysuitable volume. The formulation volume can be, for example, about 0.1mL, about 0.2 mL, about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.6mL, about 0.7 mL, about 0.8 mL, about 0.9 mL, about 1 mL, about 1.1 mL,about 1.2 mL, about 1.3 mL, about 1.4 mL, about 1.5 mL, about 1.6 mL,about 1.7 mL, about 1.8 mL, about 1.9 mL, about 2 mL, about 2.1 mL,about 2.2 mL, about 2.3 mL, about 2.4 mL, about 2.5 mL, about 2.6 mL,about 2.7 mL, about 2.8 mL, about 2.9 mL, about 3 mL, about 3.1 mL,about 3.2 mL, about 3.3 mL, about 3.4 mL, about 3.5 mL, about 3.6 mL,about 3.7 mL, about 3.8 mL, about 3.9 mL, about 4 mL, about 4.1 mL,about 4.2 mL, about 4.3 mL, about 4.4 mL, about 4.5 mL, about 4.6 mL,about 4.7 mL, about 4.8 mL, about 4.9 mL, about 5 mL, about 5.1 mL,about 5.2 mL, about 5.3 mL, about 5.4 mL, about 5.5 mL, about 5.6 mL,about 5.7 mL, about 5.8 mL, about 5.9 mL, about 6 mL, about 6.1 mL,about 6.2 mL, about 6.3 mL, about 6.4 mL, about 6.5 mL, about 6.6 mL,about 6.7 mL, about 6.8 mL, about 6.9 mL, about 7 mL, about 7.1 mL,about 7.2 mL, about 7.3 mL, about 7.4 mL, about 7.5 mL, about 7.6 mL,about 7.7 mL, about 7.8 mL, about 7.9 mL, about 8 mL, about 8.1 mL,about 8.2 mL, about 8.3 mL, about 8.4 mL, about 8.5 mL, about 8.6 mL,about 8.7 mL, about 8.8 mL, about 8.9 mL, about 9 mL, about 9.1 mL,about 9.2 mL, about 9.3 mL, about 9.4 mL, about 9.5 mL, about 9.6 mL,about 9.7 mL, about 9.8 mL, about 9.9 mL, or about 10 mL.

A therapeutically-effective amount of a compound described herein can bepresent in a composition at a concentration of, for example, about 0.1units/mL, about 0.2 units/mL, about 0.3 units/mL, about 0.4 units/mL,about 0.5 units/mL, about 0.6 units/mL, about 0.7 units/mL, about 0.8units/mL, about 0.9 units/mL, about 1 unit/mL, about 2 units/mL, about 3units/mL, about 4 units/mL, about 5 units/mL, about 6 units/mL, about 7units/mL, about 8 units/mL, about 9 units/mL, about 10 units/mL, about11 units/mL, about 12 units/mL, about 13 units/mL, about 14 units/mL,about 15 units/mL, about 16 units/mL, about 17 units/mL, about 18units/mL, about 19 units/mL, about 20 units/mL, about 21 units/mL, about22 units/mL, about 23 units/mL, about 24 units/mL about 25 units/mL,about 30 units/mL, about 35 units/mL, about 40 units/mL, about 45units/mL, or about 50 units/mL.

A therapeutically-effective amount of a compound described herein can bepresent in a composition of the invention at a mass of about, forexample, about 0.01 μg, about 0.05 μg, about 0.1 μg, about 0.15 μg,about 0.2 μg, about 0.25 μg, about 0.3 μg, about 0.35 μg, about 0.4 μg,about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg,about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 10 μg,about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about40 μg, about 45 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg,about 90 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg,about 200 μg, about 250 μg, about 300 μg, about 350 μg, about 400 μg,about 450 μg, about 500 μg, about 600 μg, about 700 μg, about 800 μg,about 900 μg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, or about 10 mg.

A therapeutically-effective amount of a compound described herein can bepresent in a composition of the invention at a concentration of, forexample, about 0.001 mg/mL, about 0.002 mg/mL, about 0.003 mg/mL, about0.004 mg/mL, about 0.005 mg/mL, about 0.006 mg/mL, about 0.007 mg/mL,about 0.008 mg/mL, about 0.009 mg/mL, about 0.01 mg/mL, about 0.02mg/mL, about 0.03 mg/mL, about 0.04 mg/mL, about 0.05 mg/mL, about 0.06mg/mL, about 0.07 mg/mL, about 0.08 mg/mL, about 0.09 mg/mL, about 0.1mg/mL, about 0.2 mg/mL, about 0.3 mg/mL, about 0.4 mg/mL, about 0.5mg/mL, about 0.6 mg/mL, about 0.7 mg/mL, about 0.8 mg/mL, about 0.9mg/mL, about 1 mg/mL, about 1.5 mg/mL, about 2 mg/mL, about 2.5 mg/mL,about 3 mg/mL, about 3.5 mg/mL, about 4 mg/mL, about 4.5 mg/mL, about 5mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL, about 9 mg/mL, orabout 10 mg/mL.

A therapeutically-effective amount of a compound described herein can bepresent in a composition of the invention at a unit of active agent/unitof active time. Non-limiting examples of therapeutically-effectiveamounts can be, for example, about 0.01 units/minute, about 0.02units/minute, about 0.03 units/minute, about 0.04 units/minute, about0.05 units/minute, about 0.06 units/minute, about 0.07 units/minute,about 0.08 units/minute, about 0.09 units/minute or about 0.1units/minute.

Pharmaceutical compositions of the invention can be formulated at anysuitable pH. The pH can be, for example, about 2, about 2.05, about 2.1,about 2.15, about 2.2, about 2.25, about 2.3, about 2.35, about 2.4,about 2.45, about 2.5, about 2.55, about 2.6, about 2.65, about 2.7,about 2.75, about 2.8, about 2.85, about 2.9, about 2.95, about 3, about3.05, about 3.1, about 3.15, about 3.2, about 3.25, about 3.3, about3.35, about 3.4, about 3.45, about 3.5, about 3.55, about 3.6, about3.65, about 3.7, about 3.75, about 3.8, about 3.85, about 3.9, about3.95, about 4, about 4.05, about 4.1, about 4.15, about 4.2, about 4.25,about 4.3, about 4.35, about 4.4, about 4.45, about 4.5, about 4.55,about 4.6, about 4.65, about 4.7, about 4.75, about 4.8, about 4.85,about 4.9, about 4.95, or about 5 pH units.

In some embodiments, the addition of an excipient can change theviscosity of a pharmaceutical composition of the invention. In someembodiments the use of an excipient can increase or decrease theviscosity of a fluid by at least 0.001 Pascal-second (Pa·s), at least0.001 Pa·s, at least 0.0009 Pa·s, at least 0.0008 Pa·s, at least 0.0007Pa·s, at least 0.0006 Pa·s, at least 0.0005 Pa·s, at least 0.0004 Pa·s,at least 0.0003 Pa·s, at least 0.0002 Pa·s, at least 0.0001 Pa·s, atleast 0.00005 Pa·s, or at least 0.00001 Pa·s.

In some embodiments, the addition of an excipient to a pharmaceuticalcomposition of the invention can increase or decrease the viscosity ofthe composition by at least 5%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or at least 99%. In some embodiments, the addition of an excipientto a pharmaceutical composition of the invention can increase ordecrease the viscosity of the composition by no greater than 5%, nogreater than 10%, no greater than 15%, no greater than 20%, no greaterthan 25%, no greater than 30%, no greater than 35%, no greater than 40%,no greater than 45%, no greater than 50%, no greater than 55%, nogreater than 60%, no greater than 65%, no greater than 70%, no greaterthan 75%, no greater than 80%, no greater than 85%, no greater than 90%,no greater than 95%, or no greater than 99%.

Any compound herein can be purified. A compound can be at least 1% pure,at least 2% pure, at least 3% pure, at least 4% pure, at least 5% pure,at least 6% pure, at least 7% pure, at least 8% pure, at least 9% pure,at least 10% pure, at least 11% pure, at least 12% pure, at least 13%pure, at least 14% pure, at least 15% pure, at least 16% pure, at least17% pure, at least 18% pure, at least 19% pure, at least 20% pure, atleast 21% pure, at least 22% pure, at least 23% pure, at least 24% pure,at least 25% pure, at least 26% pure, at least 27% pure, at least 28%pure, at least 29% pure, at least 30% pure, at least 31% pure, at least32% pure, at least 33% pure, at least 34% pure, at least 35% pure, atleast 36% pure, at least 37% pure, at least 38% pure, at least 39% pure,at least 40% pure, at least 41% pure, at least 42% pure, at least 43%pure, at least 44% pure, at least 45% pure, at least 46% pure, at least47% pure, at least 48% pure, at least 49% pure, at least 50% pure, atleast 51% pure, at least 52% pure, at least 53% pure, at least 54% pure,at least 55% pure, at least 56% pure, at least 57% pure, at least 58%pure, at least 59% pure, at least 60% pure, at least 61% pure, at least62% pure, at least 63% pure, at least 64% pure, at least 65% pure, atleast 66% pure, at least 67% pure, at least 68% pure, at least 69% pure,at least 70% pure, at least 71% pure, at least 72% pure, at least 73%pure, at least 74% pure, at least 75% pure, at least 76% pure, at least77% pure, at least 78% pure, at least 79% pure, at least 80% pure, atleast 81% pure, at least 82% pure, at least 83% pure, at least 84% pure,at least 85% pure, at least 86% pure, at least 87% pure, at least 88%pure, at least 89% pure, at least 90% pure, at least 91% pure, at least92% pure, at least 93% pure, at least 94% pure, at least 95% pure, atleast 96% pure, at least 97% pure, at least 98% pure, at least 99% pure,at least 99.1% pure, at least 99.2% pure, at least 99.3% pure, at least99.4% pure, at least 99.5% pure, at least 99.6% pure, at least 99.7%pure, at least 99.8% pure, or at least 99.9% pure.

Compositions of the invention can be packaged as a kit. In someembodiments, a kit includes written instructions on the administrationor use of the composition. The written material can be, for example, alabel. The written material can suggest conditions methods ofadministration. The instructions provide the subject and the supervisingphysician with the best guidance for achieving the optimal clinicaloutcome from the administration of the therapy. In some embodiments, thelabel can be approved by a regulatory agency, for example the U.S. Foodand Drug Administration (FDA), the European Medicines Agency (EMA), orother regulatory agencies.

Pharmaceutically-Acceptable Excipients.

Non-limiting examples of pharmaceutically-acceptable excipients can befound, for example, in Remington: The Science and Practice of Pharmacy,Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, JohnE., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa. 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical DosageForms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &Wilkins 1999), each of which is incorporated by reference in itsentirety.

In some embodiments, the pharmaceutical composition provided hereincomprises a sugar as an excipient. Non-limiting examples of sugarsinclude trehalose, sucrose, glucose, lactose, galactose, glyceraldehyde,fructose, dextrose, maltose, xylose, mannose, maltodextrin, starch,cellulose, lactulose, cellobiose, mannobiose, and combinations thereof.

In some embodiments, the pharmaceutical composition provided hereincomprises a buffer as an excipient. Non-limiting examples of buffersinclude potassium phosphate, sodium phosphate, saline sodium citratebuffer (SSC), acetate, saline, physiological saline, phosphate buffersaline (PBS), 4-2-hydroxyethyl-1-piperazineethanesulfonic acid buffer(HEPES), 3-(N-morpholino)propanesulfonic acid buffer (MOPS), andpiperazine-N,N′-bis(2-ethanesulfonic acid) buffer (PIPES), orcombinations thereof.

In some embodiments, a pharmaceutical composition of the inventioncomprises a source of divalent metal ions as an excipient. A metal canbe in elemental form, a metal atom, or a metal ion. Non-limitingexamples of metals include transition metals, main group metals, andmetals of Group 1, Group 2, Group 3, Group 4, Group 5, Group 6, Group 7,Group 8, Group 9, Group 10, Group 11, Group 12, Group 13, Group 14, andGroup 15 of the Periodic Table. Non-limiting examples of metals includelithium, sodium, potassium, cesium, magnesium, calcium, strontium,scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel,copper, zinc, yttrium, zirconium, niobium, molybdenum, palladium,silver, cadmium, tungsten, rhenium, osmium, iridium, platinum, gold,mercury, cerium, and samarium.

In some embodiments, the pharmaceutical composition provided hereincomprises an alcohol as an excipient. Non-limiting examples of alcoholsinclude ethanol, propylene glycol, glycerol, polyethylene glycol,chrlorobutanol, isopropanol, xylitol, sorbitol, maltitol, erythritol,threitol, arabitol, ribitol, mannitol, galactilol, fucitol, lactitol,and combinations thereof.

Pharmaceutical preparations can be formulated with polyethylene glycol(PEG). PEGs with molecular weights ranging from about 300 g/mol to about10,000,000 g/mol can be used. Non-limiting examples of PEGs include PEG200, PEG 300, PEG 400, PEG 540, PEG 550, PEG 600, PEG 1000, PEG 1450,PEG 1500, PEG 2000, PEG 3000, PEG 3350, PEG 4000, PEG 4600, PEG 6000,PEG 8000, PEG 10,000, and PEG 20,000.

Further excipients that can be used in a composition of the inventioninclude, for example, benzalkonium chloride, benzethonium chloride,benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene,dehydroacetic acid, ethylenediamine, ethyl vanillin, glycerin,hypophosphorous acid, phenol, phenylethyl alcohol, phenylmercuricnitrate, potassium benzoate, potassium metabisulfite, potassium sorbate,sodium bisulfite, sodium metabisulfite, sorbic acid, thimerasol, aceticacid, aluminum monostearate, boric acid, calcium hydroxide, calciumstearate, calcium sulfate, calcium tetrachloride, cellulose acetatepthalate, microcrystalline celluose, chloroform, citric acid, edeticacid, and ethylcellulose.

In some embodiments, the pharmaceutical composition provided hereincomprises an aprotic solvent as an excipient. Non-limiting examples ofaprotic solvents include perfluorohexane, α,α,α-trifluorotoluene,pentane, hexane, cyclohexane, methylcyclohexane, decalin, dioxane,carbon tetrachloride, freon-11, benzene, toluene, carbon disulfide,diisopropyl ether, diethyl ether, t-butyl methyl ether, ethyl acetate,1,2-dimethoxyethane, 2-methoxyethyl ether, tetrahydrofuran, methylenechloride, pyridine, 2-butanone, acetone, N-methylpyrrolidinone,nitromethane, dimethylformamide, acetonitrile, sulfolane, dimethylsulfoxide, and propylene carbonate.

The amount of the excipient in a pharmaceutical composition of theinvention can be about 0.01%, about 0.02%, about 0.03%, about 0.04%,about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 6%,about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%,about 70%, about 80%, about 90%, about 100%, about 200%, about 300%,about 400%, about 500%, about 600%, about 700%, about 800%, about 900%,or about 1000% by mass of the vasopressin in the pharmaceuticalcomposition.

The amount of the excipient in a pharmaceutical composition of theinvention can be about 0.01%, about 0.02%, about 0.03%, about 0.04%,about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 6%,about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, about 99%, or about 100%, mass or by volume of the unitdosage form.

The ratio of vasopressin to an excipient in a pharmaceutical compositionof the invention can be about 100:about 1, about 95:about 1, about90:about 1, about 85:about 1, about 80:about 1, about 75:about 1, about70:about 1, about 65:about 1, about 60:about 1, about 55:about 1, about50:about 1, about 45:about 1, about 40:about 1, about 35:about 1 about30:about 1, about 25:about 1, about 20:about 1, about 15:about 1, about10:about 1, about 9:about 1, about 8:about 1, about 7:about 1, about6:about 1, about 5:about 1, about 4:about 1, about 3:about 1, about2:about 1, about 1:about 1, about 1:about 2, about 1:about 3, about1:about 4, about 1: about 5, about 1:about 6, about 1:about 7, about1:about 8, about 1:about 9, or about 1:about 10.

Pharmaceutically-Acceptable Salts.

The invention provides the use of pharmaceutically-acceptable salts ofany therapeutic compound described herein. Pharmaceutically-acceptablesalts include, for example, acid-addition salts and base-addition salts.The acid that is added to the compound to form an acid-addition salt canbe an organic acid or an inorganic acid. A base that is added to thecompound to form a base-addition salt can be an organic base or aninorganic base. In some embodiments, a pharmaceutically-acceptable saltis a metal salt. In some embodiments, a pharmaceutically-acceptable saltis an ammonium salt.

Metal salts can arise from the addition of an inorganic base to acompound of the invention. The inorganic base consists of a metal cationpaired with a basic counterion, such as, for example, hydroxide,carbonate, bicarbonate, or phosphate. The metal can be an alkali metal,alkaline earth metal, transition metal, or main group metal. In someembodiments, the metal is lithium, sodium, potassium, cesium, cerium,magnesium, manganese, iron, calcium, strontium, cobalt, titanium,aluminum, copper, cadmium, or zinc.

In some embodiments, a metal salt is a lithium salt, a sodium salt, apotassium salt, a cesium salt, a cerium salt, a magnesium salt, amanganese salt, an iron salt, a calcium salt, a strontium salt, a cobaltsalt, a titanium salt, an aluminum salt, a copper salt, a cadmium salt,or a zinc salt.

Ammonium salts can arise from the addition of ammonia or an organicamine to a compound of the invention. In some embodiments, the organicamine is triethyl amine, diisopropyl amine, ethanol amine, diethanolamine, triethanol amine, morpholine, N-methylmorpholine, piperidine,N-methylpiperidine, N-ethylpiperidine, dibenzylamine, piperazine,pyridine, pyrrazole, pipyrrazole, imidazole, pyrazine, or pipyrazine.

In some embodiments, an ammonium salt is a triethyl amine salt, adiisopropyl amine salt, an ethanol amine salt, a diethanol amine salt, atriethanol amine salt, a morpholine salt, an N-methylmorpholine salt, apiperidine salt, an N-methylpiperidine salt, an N-ethylpiperidine salt,a dibenzylamine salt, a piperazine salt, a pyridine salt, a pyrrazolesalt, a pipyrrazole salt, an imidazole salt, a pyrazine salt, or apipyrazine salt.

Acid addition salts can arise from the addition of an acid to a compoundof the invention. In some embodiments, the acid is organic. In someembodiments, the acid is inorganic. In some embodiments, the acid ishydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid,nitrous acid, sulfuric acid, sulfurous acid, a phosphoric acid,isonicotinic acid, lactic acid, salicylic acid, tartaric acid, ascorbicacid, gentisinic acid, gluconic acid, glucaronic acid, saccaric acid,formic acid, benzoic acid, glutamic acid, pantothenic acid, acetic acid,propionic acid, butyric acid, fumaric acid, succinic acid,methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid, citric acid, oxalic acid, or maleic acid.

In some embodiments, the salt is a hydrochloride salt, a hydrobromidesalt, a hydroiodide salt, a nitrate salt, a nitrite salt, a sulfatesalt, a sulfite salt, a phosphate salt, isonicotinate salt, a lactatesalt, a salicylate salt, a tartrate salt, an ascorbate salt, agentisinate salt, a gluconate salt, a glucaronate salt, a saccaratesalt, a formate salt, a benzoate salt, a glutamate salt, a pantothenatesalt, an acetate salt, a propionate salt, a butyrate salt, a fumaratesalt, a succinate salt, a methanesulfonate (mesylate) salt, anethanesulfonate salt, a benzenesulfonate salt, a p-toluenesulfonatesalt, a citrate salt, an oxalate salt, or a maleate salt.

Peptide Sequence.

As used herein, the abbreviations for the L-enantiomeric andD-enantiomeric amino acids are as follows: alanine (A, Ala); arginine(R, Arg); asparagine (N, Asn); aspartic acid (D, Asp); cysteine (C,Cys); glutamic acid (E, Glu); glutamine (Q, Gln); glycine (G, Gly);histidine (H, His); isoleucine (I, Ile); leucine (L, Leu); lysine (K,Lys); methionine (M, Met); phenylalanine (F, Phe); proline (P, Pro);serine (S, Ser); threonine (T, Thr); tryptophan (W, Trp); tyrosine (Y,Tyr); valine (V, Val). In some embodiments, the amino acid is aL-enantiomer. In some embodiments, the amino acid is a D-enantiomer.

A peptide of the disclosure can have about 5%, about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, orabout 100% amino acid sequence homology to SEQ ID NO. 1.

In some embodiments, a pharmaceutical composition of the inventioncomprises one or a plurality of peptides having about 80% to about 90%sequence homology to SEQ ID NO. 1, about 88% to about 90% sequencehomology to SEQ ID NO. 1 or 88% to 90% sequence homology to SEQ IDNO. 1. In some embodiments, a pharmaceutical composition of theinvention comprises vasopression and one or more of a second, third,fourth, fifth, sixth, seventh, eighth, ninth, and tenth, peptide.

The ratio of vasopressin to another peptide in a pharmaceuticalcomposition of the invention can be, for example, about 1000:about 1,about 990:about 1, about 980:about 1, about 970:about 1, about 960:about1, about 950:about 1, about 800:about 1, about 700:about 1, about 600:1,about 500:about 1, about 400:about 1, about 300:about 1, about 200:about1, about 100:about 1, about 95:about 1, about 90:about 1, about 85:about1, about 80:about 1, about 75:about 1, about 70:about 1, about 65:about1, about 60:about 1, about 55:about 1, about 50:about 1, about 45:about1, about 40:about 1, about 35:about 1, about 30:about 1, about 25:about1, about 20:about 1, about 19:about 1, about 18:about 1, about 17:about1, about 16:about 1, about 15:about 1, about 14:about 1, about 13:about1, about 12:about 1, about 11:about 1, or about 10:about 1.

The amount of another peptide in a composition of the invention can be,for example, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%,about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%,about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%,about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%,about 9.5%, about 10%, about 11%, about 12%, about 13%, about 14%, about15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or about 100% by mass of vasopressin.

Non-limiting examples of methods that can be used to identify peptidesof the invention include high-performance liquid chromatography (HPLC),mass spectrometry (MS), Matrix Assisted Laser Desorption IonizationTime-of-Flight (MALDI-TOF), electrospray ionization Time-of-flight(ESI-TOF), gas chromatography-mass spectrometry (GC-MS), liquidchromatography-mass spectrometry (LC-MS), and two-dimensional gelelectrophoresis.

HPLC can be used to identify peptides using high pressure to separatecomponents of a mixture through a packed column of solid adsorbentmaterial, denoted the stationary phase. The sample components caninteract differently with the column based upon the pressure applied tothe column, material used in stationary phase, size of particles used inthe stationary phase, the composition of the solvent used in the column,and the temperature of the column. The interaction between the samplecomponents and the stationary phase can affect the time required for acomponent of the sample to move through the column. The time requiredfor component to travel through the column from injection point toelution is known as the retention time.

Upon elution from the column, the eluted component can be detected usinga UV detector attached to the column. The wavelength of light at whichthe component is detected, in combination with the component's retentiontime, can be used to identify the component. Further, the peak displayedby the detector can be used to determine the quantity of the componentpresent in the initial sample. Wavelengths of light that can be used todetect sample components include, for example, about 200 nM, about 225nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350nm, about 375 nm, and about 400 nm.

Mass spectrometry (MS) can also be used to identify peptides of theinvention. To prepare samples for MS analysis, the samples, containingthe proteins of interest, are digested by proteolytic enzymes intosmaller peptides. The enzymes used for cleavage can be, for example,trypsin, chymotrypsin, glutamyl endopeptidase, Lys-C, and pepsin. Thesamples can be injected into a mass spectrometer. Upon injection, all ormost of the peptides can be ionized and detected as ions on a spectrumaccording to the mass to charge ratio created upon ionization. The massto charge ratio can then be used to determine the amino acid residuespresent in the sample.

The present disclosure provides several embodiments of pharmaceuticalformulations that provide advantages in stability, administration,efficacy, and modulation of formulation viscosity. Any embodimentsdisclosed herein can be used in conjunction or individually. Forexample, any pharmaceutically-acceptable excipient, method, technique,solvent, compound, or peptide disclosed herein can be used together withany other pharmaceutically-acceptable excipient, method, technique,solvent, compound, or peptide disclosed herein to achieve anytherapeutic result. Compounds, excipients, and other formulationcomponents can be present at any amount, ratio, or percentage disclosedherein in any such formulation, and any such combination can be usedtherapeutically for any purpose described herein and to provide anyviscosity described herein.

EXAMPLES Example 1 Impurities of Vasopressin as Detected by HPLC

To analyze degradation products of vasopressin that can be present in anillustrative formulation of vasopressin, gradient HPLC was performed toseparate vasopressin from related peptides and formulation components.TABLE 2 below depicts the results of the experiment detailing thechemical formula, relative retention time (RRT), molar mass, andstructure of vasopressin and detected impurities.

Vasopressin was detected in the eluent using UV absorbance. Theconcentration of vasopressin in the sample was determined by theexternal standard method, where the peak area of vasopressin in sampleinjections was compared to the peak area of vasopressin referencestandards in a solution of known concentration. The concentrations ofrelated peptide impurities in the sample were also determined using theexternal standard method, using the vasopressin reference standard peakarea and a unit relative response factor. An impurities marker solutionwas used to determine the relative retention times of identified relatedpeptides at the time of analysis.

Experimental conditions are summarized in TABLE 2 below.

TABLE 2 a Column YMC-Pack ODS-AM, 3 μm, 120 Å pore, 4.6 × 100 mm bColumn Temperature 25° C. Flow Rate 1.0 mL/min Detector 215 nm Note: ForIdentification a Diode Array Detector (DAD) was used with the range of200-400 nm. Injection Volume 100 μL Run time 55 minutes AutosamplerVials Polypropylene vials Pump (gradient) Time (min) % A % B Flow 0 9010 1.0 40 50 50 1.0 45 50 50 1.0 46 90 10 1.0 55 90 10 1.0

The diluent used for the present experiment was 0.25% v/v Acetic Acid,which was prepared by transferring 2.5 mL of glacial acetic acid into a1-L volumetric flask containing 500 mL of water. The solution wasdiluted to the desired volume with water.

Phosphate buffer at pH 3.0 was used for mobile phase A. The buffer wasprepared by weighing approximately 15.6 g of sodium phosphate monobasicmonohydrate into a beaker. 1000 mL of water was added, and mixed well.The pH was adjusted to 3.0 with phosphoric acid. The buffer was filteredthrough a 0.45 μm membrane filter under vacuum, and the volume wasadjusted as necessary.

An acetonitrile:water (50:50) solution was used for mobile phase B. Toprepare mobile phase B, 500 mL of acetonitrile was mixed with 500 mL ofwater.

The working standard solution contained approximately 20 units/mL ofvasopressin. The standard solution was prepared by quantitativelytransferring the entire contents of 1 vial of USP Vasopressin RS withdiluent to a 50-mL volumetric flask.

The intermediate standard solution was prepared by pipetting 0.5 mL ofthe working standard solution into a 50-mL volumetric flask.

The sensitivity solution was prepared by pipetting 5.0 mL of theintermediate standard solution into a 50-mL volumetric flask. Thesolution was diluted to the volume with Diluent and mixed well.

A second working standard solution was prepared as directed under thestandard preparation.

A portion of the vasopressin control sample was transferred to an HPLCvial and injected. The control was stable for 120 hours when stored inautosampler vials at ambient laboratory conditions.

To prepare the impurities marker solution, a 0.05% v/v acetic acidsolution was prepared by pipetting 200.0 mL of a 0.25% v/v acetic acidsolution into a 1-L volumetric flask. The solution was diluted to thedesired volume with water and mixed well.

To prepare the vasopressin impurity stock solutions, the a solution ofeach impurity was prepared in a 25 mL volumetric flask and diluted with0.05% v/v acetic acid to a concentration suitable for HPLC injection.

To prepare the MAA/H-IBA (Methacrylic Acid/α-Hydroxy-isobutyric acid)stock solution, a stock solution containing approximately 0.3 mg/mLH-IBA and 0.01 mg/mL in 0.05% v/v acetic acid was made in a 50 mLvolumetric flask.

To prepare the chlorobutanol diluent, about one gram of hydrouschlorobutanol was added to 500 mL of water. Subsequently, 0.25 mL ofacetic acid was added and the solution was stirred to dissolve thechlorobutanol.

To prepare the impurity marker solution, vasopressin powder was mixedwith the impurity stock solutions prepared above.

The solutions were diluted to volume with the chlorobutanol diluent. Thesolutions were aliquoted into individual crimp top vials and stored at2-8° C. At time of use, the solutions were removed from refrigeration(2-8° C.) and allowed to reach room temperature.

The vasopressin impurity marker solution was stable for at least 120hours when stored in auto-sampler vials at ambient laboratoryconditions. The solution was suitable for use as long as thechromatographic peaks could be identified based on comparison to thereference chromatogram.

To begin the analysis, the HPLC system was allowed to equilibrate for atleast 30 minutes using mobile phase B, followed by time 0 min gradientconditions until a stable baseline was achieved.

The diluent was injected at the beginning of the run, and had no peaksthat interfered with Vasopressin at around 18 minutes as shown in FIG.1.

A single injection of the sensitivity solution was performed, whereinthe signal-to-noise ratio of the Vasopressin was greater than or equalto ten as shown in FIG. 2.

A single injection of the impurities marker solution was then made. Thelabeled impurities in the reference chromatogram were identified in thechromatogram of the marker solution based on their elution order andapproximate retention times shown in FIG. 3 and FIG. 4. FIG. 4 is azoomed in chromatograph of FIG. 3 showing the peaks that eluted between15 and 30 minutes. The nomenclature, structure, and approximateretention times for individual identified impurities are detailed inTABLE 3.

A single injection of the working standard solution was made to ensurethat the tailing factor of the vasopressin peak was less than or equalto about 2.0 as shown in FIG. 5.

A total of five replicate injections of the working standard solutionwere made to ensure that the relative standard deviation (% RSD) of thefive replicate vasopressin peak areas was not more than 2.0%.

Two replicate injections of the check standard preparation were toconfirm that the check standard conformity was 99.0%-101.0%. Oneinjection of the control sample was made to confirm that the assay ofthe control sample met the control limits established for the sample.

Then, one injection of the working standard solution was made.

Following the steps above done to confirm system suitability, a singleinjection of each sample preparation was made. The chromatograms wereanalyzed to determine the vasopressin and impurity peak areas. Thechromatogram is depicted in FIG. 6.

The working standard solution was injected after 1 to 4 sampleinjections, and the bracketing standard peak areas were averaged for usein the calculations to determine peak areas of vasopressin andassociated impurities.

The relative standard deviation (% RSD) of vasopressin peak areas forthe six injections of working standard solution was calculated byincluding the initial five injections from the system suitability stepsabove and each of the subsequent interspersed working standard solutioninjections. The calculations were done to ensure that each of the % RSDwere not more than 2.0%.

The retention time of the major peak in the chromatogram of the samplepreparation corresponded to that of the vasopressin peak in the workingstandard solution injection that preceded the sample preparationinjection.

The UV spectrum (200-400 nm) of the main peak in the chromatogram of thesample preparation compared to the UV spectrum of vasopressin in theworking standard preparation. FIG. 7 depicts a UV spectrum of avasopressin sample and FIG. 8 depicts a UV spectrum of vasopressinstandard.

To calculate the vasopressin units/mL, the following formula was used:

${{Vasopressin}\mspace{14mu} {units}\text{/}{mL}} = {\frac{R_{U}}{R_{S}} \times {Conc}\mspace{14mu} {STD}}$

where:

-   -   R_(U)=Vasopressin peak area response of Sample preparation.    -   R_(S)=average vasopressin peak area response of bracketing        standards.    -   Conc STD=concentration of the vasopressin standard in units/mL

To identify the impurities, the % Impurity and identity for identifiedimpurities (TABLE 3) that are were greater than or equal to 0.10% werereported. Impurities were truncated to 3 decimal places and then roundedto 2 decimal places, unless otherwise specified.

The impurities were calculated using the formula below:

${\% \mspace{14mu} {impurity}} = {\frac{R_{I}}{R_{S}} \times \frac{{Conc}\mspace{14mu} {STD}}{20\mspace{14mu} U\text{/}{mL}} \times 100\%}$

where:

-   -   R_(I)=Peak area response for the impurity    -   20 U/mL=Label content of vasopressin

TABLE 3 below details the chemical formula, relative retention time (RRTin minutes), molar mass, and structure of vasopressin and detectedimpurities.

TABLE 3 Name Formula Appr. RRT Molar Mass (g) Vasopressin C₄₆H₆₅N₁₅O₁₂S₂1.00 1084.23 (Arginine Vasopressin, AVP) CYFQNCPRG-NH₂ SEQ ID NO.: 1(disulfide bridge between cys residues) Gly9-vasopressin C₄₆H₆₄N₁₄O₁₃S₂1.07 1085.22 (Gly9-AVP) CYFQNCPRG SEQ ID NO.: 2 (disulfide bridgebetween cys residues) Asp5-vasopressin C₄₆H₆₄N₁₄O₁₃S₂ 1.09 1085.22(Asp5-AVP) CYFQDCPRG-NH₂ SEQ ID NO.: 3 (disulfide bridge between cysresidues) Glu4-vasopressin C₄₆H₆₄N₁₄O₁₃S₂ 1.12 1085.22 (Glu4-AVP)CYFENCPRG-NH₂ SEQ ID NO.: 4 (disulfide bridge between cys residues)Acetyl-vasopressin C₄₈H₆₇N₁₅O₁₃S₂ 1.45 1126.27 (Acetyl-AVP)Ac-CYFQNCPRG-NH₂ SEQ ID NO.: 7 (disulfide bridge between cys residues)D-Asn-vasopressin C₄₆H₆₅N₁₅O₁₂S₂ 0.97 1084.23 (DAsn-AVP)CYFQ(D-Asn)CPRG-NH₂ SEQ ID NO.: 10 (disulfide bridge between cysresidues) Dimeric-vasopressin C₉₂H₁₃₀N₃₀O₂₄S₄ 1.22 2168.46 (Dimer-AVP)(monomers cross linked by disulfide bridges)

Example 2 Investigation of pH

To determine a possible pH for a vasopressin formulation with good shelflife, vasopressin formulations were prepared in 10 mM citrate bufferdiluted in isotonic saline across a range of pH. Stability was assessedvia HPLC as in EXAMPLE 1 after incubation of the formulations at 60° C.for one week. FIG. 9 illustrates the results of the experiment. Thegreatest level of stability was observed at pH 3.5. At pH 3.5, thepercent label claim (% LC) of vasopressin was highest, and theproportion of total impurities was lowest.

Example 3 Effect of Peptide Stabilizers on Vasopressin Formulation

To observe the effect of stabilizers on the degradation of vasopressin,a series of peptide stabilizers were added to a vasopressin formulationas detailed in TABLE 4. Stability of vasopressin was assessed via HPLCafter incubation of the formulations at 60° C. for one week.

TABLE 4 PEG Poloxamer n-Methylpyrrolidone Ethanol 400 Glycerol 188HPbCD^(a) (NMP)  1%  1%  1%  1%  1%  1% 10% 10% 10% 10% 10% 10%^(a)Hydroxypropyl beta-Cyclodextrin

FIG. 10 illustrates the stability of vasopressin in terms of % labelclaim at varying concentrations of stabilizer. The results indicate thatthe tested stabilizers provided a greater stabilizing effect at 1%concentration than at 10%. Also, in several cases the stabilizationeffect was about 5% to about 10% greater than that observed in theexperiments of EXAMPLE 2.

Example 4 Effect of Buffer and Divalent Metals on VasopressinFormulation

To determine whether different combinations of buffers and use ofdivalent metals affect vasopressin stability, vasopressin formulationswith varying concentrations of citrate and acetate buffers and variableconcentrations of calcium, magnesium, and zinc ions were prepared.Solutions of 0 mM, 10 mM, 20 mM, and 80 mM calcium, magnesium, and zincwere prepared and each was combined with 1 mM or 10 mM of citrate oracetate buffers to test vasopressin stability.

The tested combinations provided vasopressin stability comparable tothat of a vasopressin formulation lacking buffers and divalent metals.However, that the addition of divalent metal ions was able to counteractthe degradation of vasopressin caused by the use of a citrate buffer.

Example 5 Effect of Non-Aqueous Solvent Formulations on VasopressinStability

Several solvents were used to prepare vasopressin formulations to assessvasopressin stability. The formulations were prepared at 400 μg/mL andstability was tested via HPLC after incubation at 60° C. for one week,40° C. for four weeks, and 25° C. for four weeks. The examined solventsincluded water, DMSO, propylene glycol, PEG300, NMP, glycerol, andethanol. None of the tested solvents were able to increase the stabilityof vasopressin in solution in comparison to an aqueous formulationlacking a cosolvent.

Example 6 Illustrative Formulations for Assessment of VasopressinStability

An aqueous formulation of vasopressin is prepared using 10% trehalose,1% sucrose, or 5% NaCl and incubated at 60° C. for one week, at whichpoint stability of vasopressin is assessed using HPLC.

A formulation containing 50 units of vasopressin is lyophilized. Thelyophilate is reconstituted with water and either 100 mg of sucrose or100 mg of lactose, and the stability of vasopressin is tested via HPLCafter incubation at 60° C. for one week.

Co-solvents are added to a vasopressin solution to assess vasopressinstability. 95% solvent/5% 20 mM acetate buffer solutions are preparedusing propylene glycol, DMSO, PEG300, NMP, glycerol, and glycerol:NMP(1:1), and used to create formulations of vasopressin. The stability ofvasopressin is tested after incubation at 60° C. for one week.

Amino acid and phosphate buffers are tested with vasopressin to assessvasopressin stability. Buffers of 10 mM glycine, aspartate, phosphateare prepared at pH 3.5 and 3.8 and used to create formulations ofvasopressin. The stability of vasopressin is tested after incubation at60° C. for one week.

A vasopressin formulation in 10% polyvinylpyrrolidone is prepared toassess vasopressin stability. The stability of vasopressin will betested after incubation at 60° C. for one week.

A vasopressin formulation that contains 0.9% saline, 10 mM acetatebuffer, 0.2 unit/mL API/mL in 100 mL of total volume is prepared. The pHof the solution is varied from pH 3.5-3.8 to test the stability ofvasopressin.

A vasopressin formulation in about 50% to about 80% DMSO (for example,about 80%), about 20% to about 50% ethyl acetate (for example, about20%), and about 5% to about 30% polyvinylpyrrolidone (PVP) (for example,about 10% by mass of the formulation) is prepared to assess vasopressinstability. PVP K12 and PVP K17 are each independently tested in theformulation. The stability of vasopressin is tested after incubation at60° C. for one week.

A vasopressin formulation in about 70% to about 95% ethyl acetate, andabout 5% to about 30% PVP is prepared to assess vasopressin stability.PVP K12 and PVP K17 are each independently tested in the formulation.The stability of vasopressin is tested after incubation at 60° C. forone week.

A vasopressin formulation in 90% DMSO and 10% PVP is prepared to testvasopressin stability. PVP K12 and PVP K17 are each independently testedin the formulation. The stability of vasopressin is tested afterincubation at 60° C. for one week.

Example 7 Illustrative Vasopressin Formulation for Clinical Use

A formulation for vasopressin that can be used in the clinic is detailedin TABLE 5 below:

TABLE 5 Ingredient Function Amount (per mL) Vasopressin, USP ActiveIngredient 20 Units (~0.04 mg) Chlorobutanol, Preservative 5.0 mgHydrous NF Acetic Acid, NF pH Adjustment To pH 3.4-3.6 (~0.22 mg) Waterfor injection, Diluent QS USP/EP

Example 8 Illustrative Regimen for Therapeutic Use of a VasopressinFormulation

Vasopressin is indicated to increase blood pressure in adults withvasodilatory shock (for example, adults who are post-cardiotomy orseptic) who remain hypotensive despite fluids and catecholamines.

Preparation and Use of Vasopressin.

Vasopressin is supplied in a carton of 25 multi-dose vials eachcontaining 1 mL vasopressin at 20 units/mL.

Vasopressin is stored between 15° C. and 25° C. (59° F. and 77° F.), andis not frozen.

Vials of vasopressin are to be discarded 48 hours after first puncture.

Vasopressin is prepared according to TABLE 6 below:

TABLE 6 Mix Fluid Restriction? Final Concentration Vasopressin DiluentNo 0.1 units/mL 2.5 mL (50 units) 500 mL Yes  1 unit/mL   5 mL (100units) 100 mL

Vasopressin is diluted in normal saline (0.9% sodium chloride) or 5%dextrose in water (D5W) prior to use to either 0.1 units/mL or 1 unit/mLfor intravenous administration. Unused diluted solution is discardedafter 18 hours at room temperature or after 24 hours underrefrigeration.

Diluted vasopressin should be inspected for particulate matter anddiscoloration prior to use whenever solution and container permit.

The goal of treatment with vasopressin is optimization of perfusion tocritical organs, but aggressive treatment can compromise perfusion oforgans, like the gastrointestinal tract, for which function is difficultto monitor. Titration of vasopressin to the lowest dose compatible witha clinically-acceptable response is recommended.

For post-cardiotomy shock, a dose of 0.03 units/minute is used as astarting point. For septic shock, a dose of 0.01 units/minute isrecommended. If the target blood pressure response is not achieved,titrate up by 0.005 units/minute at 10- to 15-minute intervals. Themaximum dose for post-cardiotomy shock is 0.1 units/minute and forseptic shock 0.07 units/minute. After target blood pressure has beenmaintained for 8 hours without the use of catecholamines, tapervasopressin by 0.005 units/minute every hour as tolerated to maintaintarget blood pressure. Vasopressin is provided at 20 units per mL ofdiluent, which is packaged as 1 mL of vasopressin per vial, and isdiluted prior to administration.

Contraindications, Adverse Reactions, and Drug-Drug Interactions.

Vasopressin is contraindicated in patients with known allergy orhypersensitivity to 8-L-arginine vasopressin or chlorobutanol.Additionally, use of vasopressin in patients with impaired cardiacresponse can worsen cardiac output.

Adverse reactions have been observed with the use of vasopressin, whichadverse reactions include bleeding/lymphatic system disorders,specifically, hemorrhagic shock, decreased platelets, intractablebleeding; cardiac disorders, specifically, right heart failure, atrialfibrillation, bradycardia, myocardial ischemia; gastrointestinaldisorders, specifically, mesenteric ischemia; hepatobiliary disorders,specifically, increased bilirubin levels; renal/urinary disorders,specifically, acute renal insufficiency; vascular disorders,specifically, distal limb ischemia; metabolic disorders, specifically,hyponatremia; and skin disorders, specifically, and ischemic lesions.

These reactions are reported voluntarily from a population of uncertainsize. Thus, reliable estimation of frequency or establishment of acausal relationship to drug exposure is unlikely.

Vasopressin has been observed to interact with other drugs. For example,use of vasopressin with catecholamines is expected to result in anadditive effect on mean arterial blood pressure and other hemodynamicparameters. Use of vasopressin with indomethacin can prolong the effectof vasopressin on cardiac index and systemic vascular resistance.Indomethacin more than doubles the time to offset for vasopressin'seffect on peripheral vascular resistance and cardiac output in healthysubjects.

Further, use of vasopressin with ganglionic blocking agents can increasethe effect of vasopressin on mean arterial blood pressure. Theganglionic blocking agent tetra-ethylammonium increases the pressoreffect of vasopressin by 20% in healthy subjects.

Use of vasopressin with furosemide increases the effect of vasopressinon osmolar clearance and urine flow. Furosemide increases osmolarclearance 4-fold and urine flow 9-fold when co-administered withexogenous vasopressin in healthy subjects.

Use of vasopressin with drugs suspected of causing SIADH (Syndrome ofinappropriate antidiuretic hormone secretion), for example, SSRIs,tricyclic antidepressants, haloperidol, chlorpropamide, enalapril,methyldopa, pentamidine, vincristine, cyclophosphamide, ifosfamide, andfelbamate can increase the pressor effect in addition to theantidiuretic effect of vasopressin. Additionally, use of vasopressinwith drugs suspected of causing diabetes insipidus for example,demeclocycline, lithium, foscarnet, and clozapine can decrease thepressor effect in addition to the antidiuretic effect of vasopressin.

Halothane, morphine, fentanyl, alfentanyl and sufentanyl do not impactexposure to endogenous vasopressin.

Use of Vasopressin in Specific Populations.

Vasopressin is a Category C drug for pregnancy.

Due to a spillover into the blood of placental vasopressinase, theclearance of exogenous and endogenous vasopressin increases graduallyover the course of a pregnancy. During the first trimester of pregnancythe clearance is only slightly increased. However, by the thirdtrimester the clearance of vasopressin is increased about 4-fold and atterm up to 5-fold. Due to the increased clearance of vasopressin in thesecond and third trimester, the dose of vasopressin can be up-titratedto doses exceeding 0.1 units/minute in post-cardiotomy shock and 0.07units/minute in septic shock. Vasopressin can produce tonic uterinecontractions that could threaten the continuation of pregnancy. Afterdelivery, the clearance of vasopressin returns to preconception levels.

Overdosage.

Overdosage with vasopressin can be expected to manifest as a consequenceof vasoconstriction of various vascular beds, for example, theperipheral, mesenteric, and coronary vascular beds, and as hyponatremia.In addition, overdosage of vasopressin can lead less commonly toventricular tachyarrhythmias, including Torsade de Pointes,rhabdomyolysis, and non-specific gastrointestinal symptoms. Directeffects of vasopressin overdose can resolve within minutes of withdrawalof treatment.

Pharmacology of Vasopressin.

Vasopressin is a polypeptide hormone that causes contraction of vascularand other smooth muscles and antidiuresis, which can be formulated as asterile, aqueous solution of synthetic arginine vasopressin forintravenous administration. The 1 mL solution contains vasopressin 20units/mL, chlorobutanol, NF 0.5% as a preservative, and water forinjection, USP adjusted with acetic acid to pH 3.4-3.6.

The chemical name of vasopressin is Cyclo (1-6)L-Cysteinyl-L-Tyrosyl-L-Phenylalanyl-L-Glutaminyl-L-Asparaginyl-L-Cysteinyl-L-Prolyl-L-Arginyl-L-Glycinamide.Vasopressin is a white to off-white amorphous powder, freely soluble inwater. The structural formula of vasopressin is:

-   -   SEQ ID NO.: 1        Molecular Formula: C₄₆H₆₅N₁₅O₁₂S₂; Molecular Weight: 1084.23

One mg of vasopressin is equivalent to 530 units.

The vasoconstrictive effects of vasopressin are mediated by vascular V1receptors. Vascular V1 receptors are directly coupled to phopholipase C,resulting in release of calcium, leading to vasoconstriction. Inaddition, vasopressin stimulates antidiuresis via stimulation of V2receptors which are coupled to adenyl cyclase.

At therapeutic doses, exogenous vasopressin elicits a vasoconstrictiveeffect in most vascular beds including the splanchnic, renal, andcutaneous circulation. In addition, vasopressin at pressor dosestriggers contractions of smooth muscles in the gastrointestinal tractmediated by muscular V1-receptors and release of prolactin and ACTH viaV3 receptors. At lower concentrations typical for the antidiuretichormone, vasopressin inhibits water diuresis via renal V2 receptors. Inpatients with vasodilatory shock, vasopressin in therapeutic dosesincreases systemic vascular resistance and mean arterial blood pressureand reduces the dose requirements for norepinephrine.

Vasopressin tends to decrease heart rate and cardiac output. The pressoreffect is proportional to the infusion rate of exogenous vasopressin.Onset of the pressor effect of vasopressin is rapid, and the peak effectoccurs within 15 minutes. After stopping the infusion, the pressoreffect fades within 20 minutes. There is no evidence for tachyphylaxisor tolerance to the pressor effect of vasopressin in patients.

At infusion rates used in vasodilatory shock (0.01-0.1 units/minute),the clearance of vasopressin is 9 to 25 mL/min/kg in patients withvasodilatory shock. The apparent half-life of vasopressin at theselevels is <10 minutes. Vasopressin is predominantly metabolized and onlyabout 6% of the dose is excreted unchanged in urine. Animal experimentssuggest that the metabolism of vasopressin is primarily by liver andkidney. Serine protease, carboxipeptidase and disulfide oxido-reductasecleave vasopressin at sites relevant for the pharmacological activity ofthe hormone. Thus, the generated metabolites are not expected to retainimportant pharmacological activity.

Carcinogenesis, Mutagenesis, Impairment of Fertility.

Vasopressin was found to be negative in the in vitro bacterialmutagenicity (Ames) test and the in vitro Chinese hamster ovary (CHO)cell chromosome aberration test. In mice, vasopressin can have an effecton function and fertilizing ability of spermatozoa.

Clinical Studies.

Increases in systolic and mean blood pressure following administrationof vasopressin were observed in seven studies in septic shock and eightstudies in post-cardiotomy vasodilatory shock.

Embodiments

The following non-limiting embodiments provide illustrative examples ofthe invention, but do not limit the scope of the invention.

In some embodiments, the invention provides a pharmaceutical compositioncomprising, in a unit dosage form: a) from about 0.01 mg/mL to about0.07 mg/mL of vasopressin, or a pharmaceutically-acceptable saltthereof; and b) a polymeric pharmaceutically-acceptable excipient in anamount that is from about 1% to about 10% by mass of the unit dosageform or the pharmaceutically-acceptable salt thereof, wherein the unitdosage form exhibits from about 5% to about 10% less degradation of thevasopressin or the pharmaceutically-acceptable salt thereof afterstorage for about 1 week at about 60° C. than does a corresponding unitdosage form, wherein the corresponding unit dosage form consistsessentially of: A) vasopressin, or a pharmaceutically-acceptable saltthereof; and B) a buffer having acidic pH. In some embodiments, thepolymeric pharmaceutically-acceptable excipient comprises a polyalkyleneoxide moiety. In some embodiments, the polymericpharmaceutically-acceptable excipient is a polyethylene oxide. In someembodiments, the polymeric pharmaceutically-acceptable excipient is apoloxamer. In some embodiments, the unit dosage form has an amount ofthe polymeric pharmaceutically-acceptable excipient that is about 1% theamount of the vasopressin or the pharmaceutically-acceptable saltthereof. In some embodiments, the first unit dosage form exhibits about10% less degradation of the vasopressin or thepharmaceutically-acceptable salt thereof after storage for about 1 weekat about 60° C. than does the corresponding unit dosage form. In someembodiments, the unit dosage form further comprises SEQ ID NO. 2. Insome embodiments, the composition further comprises SEQ ID NO. 3. Insome embodiments, the composition further comprises SEQ ID NO. 4. Insome embodiments, the unit dosage form is an injectable of about 1 mLvolume. In some embodiments, the unit dosage form consists essentiallyof: a) about 0.04 mg/mL of vasopressin, or thepharmaceutically-acceptable salt thereof; b) the polymericpharmaceutically-acceptable excipient in an amount that is from about 1%to about 10% by mass of the vasopressin or thepharmaceutically-acceptable salt thereof; and c) a plurality ofpeptides, wherein each of the peptides has from 88% to 90% sequencehomology to the vasopressin or the pharmaceutically-acceptable saltthereof. In some embodiments, one of the plurality of peptides is SEQ IDNO.: 2. In some embodiments, one of the plurality of peptides is SEQ IDNO.:3. In some embodiments, wherein one of the plurality of peptides isSEQ ID NO.: 4. In some embodiments, the buffer has a pH of about 3.5.

1-15. (canceled)
 16. A method of increasing blood pressure in a human inneed thereof, the method comprising: a) providing a pharmaceuticalcomposition for intravenous administration comprising, in a unit dosageform: i) from about 0.01 mg/mL to about 0.07 mg/mL of vasopressin or apharmaceutically-acceptable salt thereof; chlorobutanol; acetic acid;and iv) water, wherein the unit dosage form has a pH of 3.4 to 3.6; b)storing the unit dosage form at 2-8° C.; and c) administering the unitdosage form to the human; wherein: the administration provides to thehuman from about 0.01 units of vasopressin or thepharmaceutically-acceptable salt thereof per minute to about 0.1 unitsof vasopressin or the pharmaceutically-acceptable salt thereof perminute; and the human is hypotensive wherein the unit dosage formexhibits less than about 5% degradation after storage at 2-8° C. forabout four weeks.
 17. The method of claim 16, wherein the unit dosageform further comprises SEQ ID NO.: 2 in an amount of about 0.01% afterstorage for about 4 weeks at 2-8° C.
 18. The method of claim 16, whereinthe unit dosage form further comprises SEQ ID NO.: 3 in an amount ofabout 0.01% after storage for about 4 weeks at 2-8° C.
 19. The method ofclaim 16, wherein the unit dosage form further comprises SEQ ID NO.: 4in an amount of about 0.01% after storage for about 4 weeks at 2-8° C.20. The method of claim 16, wherein the human's mean arterial bloodpressure is increased within 15 minutes of administration.
 21. Themethod of claim 16, wherein the human's hypotension is associated withvasodilatory shock.
 22. The method of claim 21, wherein the vasodilatoryshock is post-cardiotomy shock.
 23. The method of claim 22, wherein theadministration provides to the human from about 0.03 units ofvasopressin or the pharmaceutically-acceptable salt thereof per minuteto about 0.1 units of vasopressin or the pharmaceutically-acceptablesalt thereof per minute
 24. The method of claim 21, wherein thevasodilatory shock is septic shock.
 25. The method of claim 24, whereinthe administration provides to the human from about 0.01 units ofvasopressin or the pharmaceutically-acceptable salt thereof per minuteto about 0.07 units of vasopressin or the pharmaceutically-acceptablesalt thereof per minute
 26. The method of claim 16, the method furthercomprising attaining a target blood pressure in the human and continuingthe administration for a period of about 8 hours.
 27. The method ofclaim 26, the method further comprising, after the period of about 8hours, reducing the administration by about 0.005 units per minute. 28.The method of claim 16, the method further comprising reaching a targetincrease in blood pressure of the human, wherein if the target increasein blood pressure is not attained, the administration is increased byabout 0.005 units per minute at 10-15 minute intervals until the targetincrease in blood pressure is attained.
 29. The method of claim 1,wherein the unit dosage form is stored at about 5° C.
 30. The method ofclaim 1, wherein the storing the unit dosage form is for 24 hours at2-8° C. prior to administering the unit dosage form to the subject. 31.The method of claim 1, wherein the storing the unit dosage form is for48 hours at 2-8° C. prior to administering the unit dosage form to thesubject.
 32. The method of claim 1, the method further comprisingdiscarding a vial containing the unit dosage form at least 48 hoursafter a first puncture of the vial.
 33. A method of increasing bloodpressure in a human in need thereof, the method comprising: a) providinga pharmaceutical composition for intravenous administration comprising,in a unit dosage form: i) from about 0.01 mg/mL to about 0.07 mg/mL ofvasopressin or a pharmaceutically-acceptable salt thereof;chlorobutanol; acetic acid; and iv) water, wherein the unit dosage formhas a pH of 3.4 to 3.6; b) storing the unit dosage form at 2-8° C. for24 hours; and c) thereafter administering the unit dosage form to thehuman; wherein: the administration provides to the human from about 0.01units of vasopressin or the pharmaceutically-acceptable salt thereof perminute to about 0.1 units of vasopressin or thepharmaceutically-acceptable salt thereof per minute; and the human ishypotensive.